Studies during the past year have identified a previously unknown role for ascorbic acid on liver microsomal drug oxidation. Additionally, a free radical mechanism has been implicated as one process for drug oxidation. A requirement for iron, H2O2 (or O2) and ascorbic acid has again been found for microsomal drug oxidation, but it is now clear that ascorbic acid is required for optimal drug oxidation by liver microsomes from non scorbutic animals. The proposed work will extend these studies to identify the mechanism of drug oxidation (via peroxides or epoxides) through use of 180 labelled oxygen or water. Mass spectrometry will be used for product identification whereas intermediates of the reaction will be subjected to TLC analysis and carbon-hydrogen content determination. The role of ascorbic acid as a reducing agent or as a free radical initiator will be studied by use of artificial oxidation systems or by use of artificial oxidation systems or by use of liver microsomes. It has already been shown that NADPH can be replaced by ascorbic acid in the reaction mixture and that ascorbic acid (and possible other factors) from the liver 100,000 x g supernatant fraction greatly stimulates microsomal drug oxidation. This oxidation is enhanced by hepatic microsomes as compared with systems containing iron in salt or chelate form or in systems with heat inactivated microsomes. Elucidation of the precise role of ascorbic acid on drug metabolism and the molecular mechanisms underlying drug hydroxylation by liver microsomes is the primary goal of future work. Additionally, new approaches to enzyme kinetics will developed to facilitate the study of drug hydroxylation via free radical or epoxidation mechanisms.